Monitoring of the alignment in developing tissue-engineered constructs by elastic scattering spectroscopy

摘要

Elastic light scattering spectroscopy was applied to monitor the development of alignment in fibroblast-populated collagen gels. Gels were seeded with human dermal fibroblasts in rectangular moulds so uniaxial tension was generated in the central zone of the gels due to cell contraction. There was a gradual transition from a disorganised matrix with round cells to highly organised cell/collagen matrix, aligned in the direction of the principal strain developed during gel contraction (observed with light microscopy under phase contrast). Spectra of the backscattered light (320-850 nm) were acquired via an optical probe with 2.75-mm source-detector separation, positioned perpendicularly to the gel surface, at 0, 17, 24, 41, 47, 65 and 72h. Spectra were registered for light propagating along, perpendicular and at intermediate angles relative to the cell/collagen matrix alignment, at 45° intervals. Backscatter was isotropic for non-contracted gels. However, as gels contracted, anisotropy of backscatter gradually increased. This was characterised by an 'anisotropy factor', AF (500 nm), calculated as the ratio of backscatter intensities at 90° and 0° positions of the probe, at 500 nm. AF (500nm) increased from 1.2 +- 0.1 at Oh up to 2.6 +- 0.4 at 72h of contraction, with more backscatter detected perpendicular to the cell/collagen matrix alignment than in parallel direction. Thus, backscatter anisotropy allows determination of the direction of the preferential alignment and quantitative monitoring of its development during gel contraction. It is possible to use measurements of this type to quantify a proportion of oriented fibrils in the gel using modelling.